The invention relates to cancer, and more particularly to cancer suppressor genes.
Breast carcinoma is the second leading cause of cancer-related death in women of the western world. In the United States alone over 175,000 new cases are diagnosed annually. The natural history of breast cancer involves a sequential progression through defined clinical and pathologic stages starting with initially benign then atypical hyperproliferation, progressing into in situ then invasive carcinomas, and culminating in metastatic disease. Ductal carcinoma in situ (DCIS) is the precursor of invasive ductal carcinoma.
The invention is based on the identification of a human gene that, while highly expressed in normal breast tissue, is not expressed or is poorly expressed in DCIS tumors as well as breast tumors at other stages. This gene has been designated the xe2x80x9cHIN-1xe2x80x9d (High in Normal 1) gene. The inventors have also identified homologues of the human HIN-1 gene in mice and rats. Human HIN-1 is designated as hHIN-1, mouse HIN-1 as mHIN-1, and rat HIN-1 as rHIN-1. Text that refers to HIN-1 without specifying human, mouse or rat is pertinent to all three forms of HIN-1. The inventors have, in addition, identified two Drosophila genes containing sequences encoding proteins with significant homology to hHIN-1. Thus, the invention features an isolated DNA encoding an HIN-1 polypeptide, purified HIN-1 polypeptides, vectors containing the DNAs, and cells containing the vectors. In addition, the invention features a method of making an HIN-1 polypeptide, in vitro and in vivo methods of inhibiting proliferation of a cancer cell, and methods of diagnosing cancer.
More specifically, the invention features an isolated DNA containing a nucleic acid sequence encoding a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:22. The DNA can, for example, include the nucleic acid sequence designated SEQ ID NO:3 or SEQ ID NO:23. The invention also includes a vector containing: (a) a nucleic acid sequence that (i) encodes a polypeptide that inhibits proliferation of breast cancer cells, and (ii) hybridizes under highly stringent conditions to a probe consisting of a sequence that is the complement of SEQ ID NO:3; or (b) the complement of the nucleic acid sequence. Vectors of the invention can also contain any of the isolated DNAs of the invention. In the vectors, polypeptide encoding sequences can be operably linked to a transcriptional regulatory element (TRE). Also encompassed by the invention is a cell (e.g., a prokaryotic or a eukaryotic cell) comprising any vector of the invention.
Also featured by the invention is an isolated polypeptide containing: (a) a protein that inhibits proliferation of breast cancer cells and that is encoded by a nucleic acid sequence that hybridizes under highly stringent conditions to a probe that includes or is the sequence that is the complement of SEQ ID NO:3; or (b) the protein, except for one or more conservative amino acid substitutions. The polypeptide can include the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:22. Another polypeptide of the invention is an isolated polypeptide containing (a) a functional fragment of any of the above-described polypeptides; or (b) the functional fragment, except for one or more conservative amino acid substitutions. Also included in the invention is a method of making a polypeptide; the method involves culturing a cell of the invention and extracting the polypeptide from the culture. The invention also features fragments of any of the DNAs of the invention, e.g., fragments of the DNA with SEQ ID NO:3 that include nucleotides 55 and 56 of SEQ ID NO:3. The fragments of the DNAs of the invention will be at least 10 bp, 15 bp, 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 250 bp, 300 bp, 305 bp, or 309 bp long.
Another aspect of the invention is a method of inhibiting proliferation of a cancer cell. The method involves contacting the cancer cell with any of the polypeptides of the invention. The cancer cell can be, for example, a breast cancer cell. The contacting can be in vitro. Alternatively, the cancer cell can be in a mammal and the contacting in the mammal can involve administering either the polypeptide or a polynucleotide encoding the polypeptide to the mammal. Where the cancer cell is in a mammal, the method can involve: (a) providing a recombinant cell that is the progeny of a cell obtained from the mammal and has been transfected or transformed ex vivo with a nucleic acid encoding the polypeptide; and (b) administering the cell to the mammal.
Another embodiment of the invention is a method of identifying a compound that enhances inhibition of proliferation of cancer cells. The method involves: (a) providing a first and a second plurality of cancer cells; (b) combining a test compound, the first plurality of cancer cells, and any of the polypeptides of the invention; (c) combining the second plurality of cancer cells and; (d) determining the level proliferation of the first plurality of cancer cells. A decreased level of proliferation of the first plurality of cancer cells, as compared to the second plurality of cells, indicates that the test compound enhances inhibition of proliferation of cancer cells by the polypeptide.
Also featured by the invention is a method of diagnosis. The method can involve (a) providing a test cell; and b) measuring the level of expression of a HIN-1 gene in the cell. Lack of expression of the HIN-1 gene or a low level of expression of the HIN-1 gene is an indication that the test cell is a cancer cell. Expression of the HIN-1 gene can be measured as a function of the level of HIN-1 mRNA in the cell or as a function of the level of HIN-1 polypeptide in the cell.
In another aspect, the invention provides a method of diagnosis. The method involves (a) providing a test cell; and (b) determining the degree of methylation of a HIN-1 promoter region in the test cell. A high degree of methylation of the HIN-1 promoter region is an indication that the test cell is a cancer cell. The test cell can be, for example, a breast cell, a prostate cell, a pancreatic cell, or a lung cell.
The invention features an antibody that binds to any of the polypeptides of invention. The antibody can be a monoclonal antibody or a polyclonal antibody.
Also included in the invention is a method of treatment that involves identifying a patient as having cancer cells in which (a) HIN-1 gene expression is low or (b) a HIN-1 promoter region is methylated; and treating the patient with a compound that reduces methylation of the HIN-1 promoter region or with a compound that induces expression of a gene with a methylated promoter region, e.g., the HIN-1 gene.
Yet another aspect of the invention is a method of identifying a compound that replaces the function of HIN-1 in cells that do not express HIN-1. The method involves: (a) providing a first cell that does not express HIN-1; (b) providing a second cell that does express HIN-1; (c) treating the first cell and the second cell with a test compound; and (d) determining whether the test compound decreases proliferation of the first or the second cell. A compound that decreases proliferation of the first cell but not the second cell can potentially replace the function of HIN-1 in cells that do not express HIN -1.
xe2x80x9cPolypeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The invention also features HIN-1 polypeptides with conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
As used herein, xe2x80x9cfull-length HIN-1xe2x80x9d is HIN-1 with its native signal sequence.
The term xe2x80x9cisolatedxe2x80x9d polypeptide or peptide fragment as used herein refers to a polypeptide or a peptide fragment which either has no naturally-occurring counterpart or has been separated or purified from components which naturally accompany it, e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue or tumor tissue, or body fluids such as blood, serum, or urine. Typically, the polypeptide or peptide fragment is considered xe2x80x9cisolatedxe2x80x9d when it is at least 70%, by dry weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, a preparation of a polypeptide (or peptide fragment thereof) of the invention is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, the polypeptide (or the peptide fragment thereof), respectively, of the invention. Thus, for example, a preparation of polypeptide x is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, polypeptide x. Since a polypeptide that is chemically synthesized is, by its nature, separated from the components that naturally accompany it, the synthetic polypeptide is xe2x80x9cisolated.xe2x80x9d
An isolated polypeptide (or peptide fragment) of the invention can be obtained, for example, by extraction from a natural source (e.g., from tissues or bodily fluids); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis. A polypeptide that is produced in a cellular system different from the source from which it naturally originates is xe2x80x9cisolated,xe2x80x9d because it will necessarily be free of components which naturally accompany it. The degree of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
An xe2x80x9cisolated DNAxe2x80x9d is either (1) a DNA that contains sequence not identical to that of any naturally occurring sequence, or (2), in the context of a DNA with a naturally-occurring sequence (e.g., a cDNA or genomic DNA), a DNA free of at least one of the genes that flank the gene containing the DNA of interest in the genome of the organism in which the gene containing the DNA of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. The term also includes a separate molecule such as: a cDNA where the corresponding genomic DNA has introns and therefore a different sequence; a genomic fragment that lacks at least one of the flanking genes; a fragment of cDNA or genomic DNA produced by polymerase chain reaction (PCR) and that lacks at least one of the flanking genes; a restriction fragment that lacks at least one of the flanking genes; a DNA encoding a non-naturally occurring protein such as a fusion protein, mutein, or fragment of a given protein; and a nucleic acid which is a degenerate variant of a cDNA or a naturally occurring nucleic acid. In addition, it includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a non-naturally occurring fusion protein. Also included is a recombinant DNA that includes a portion of SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:20. It will be apparent from the foregoing that isolated DNA does not mean a DNA present among hundreds to millions of other DNA molecules within, for example, cDNA or genomic DNA libraries or genomic DNA restriction digests in, for example, a restriction digest reaction mixture or an electrophoretic gel slice.
As used herein, a xe2x80x9cfunctional fragmentxe2x80x9d of a HIN-1 polypeptide is a fragment of the polypeptide that is shorter than the full-length polypeptide and has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more) of the ability of the full-length polypeptide to inhibit the proliferation of a cancer cell, e.g., a breast cancer cell. Fragments of interest can be made either by recombinant, synthetic, or proteolytic digestive methods. Such fragments can then be isolated and tested for their ability to inhibit the proliferation of cancer cells as measured by [3H]-thymidine incorporation or cell counting.
As used herein, xe2x80x9coperably linkedxe2x80x9d means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
As used herein, the term xe2x80x9cantibodyxe2x80x9d refers not only to whole antibody molecules, but also to antigen-binding fragments, e.g., Fab, F(abxe2x80x2)2, Fv, and single chain Fv (ScFv) fragments. Also included are chimeric antibodies.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention, e.g., inhibiting proliferation of cancer cells, will be apparent from the following description, from the drawings and from the claims.